Developing Fluorescence-based Techniques for Biochemical Detection in Dynamic Tissue Environment

This dissertation presents two novel techniques for investigating the spatial distribution of molecules within ex vivo lymphoid tissue samples, providing valuable insights into molecular organization and cellular interactions within lymphoid environments. ‎Chapter 1 underscores the importance of spatial distribution in tissue analysis, highlighting the preservation of native organization and cell-cell interactions in tissue slices. It also addresses key challenges faced by bioanalytical chemists working with living tissue samples and discusses strategies for overcoming these challenges. ‎Chapter 2 details the development of a system to monitor oxygen consumption rates in tissue slices, demonstrating regional dynamics and the potential for comparing tissue culture conditions and responses to stimulation. In ‎Chapter 3, a novel cell-surface-based immunoassay is introduced to detect local IFN-γ cytokine concentrations in tissue samples ex vivo, offering insights into immune responses without genetic modification. This method utilizes a dual-affinity reagent to capture secreted cytokines near their source, with successful detection demonstrated in CD3 Ԑ stimulated lymph node tissue slices. ‎Chapter 4 presents an optimized slicing protocol for analyzing live human tonsil tissue, preserving its intricate architecture and enabling investigations into immune functions. This protocol demonstrates the maintenance of expected spatial organization and immune responses in tonsil slices, offering a versatile platform for studying dynamic immune responses and cellular behaviors in spatially organized tissue. Lastly, ‎Chapter 5 explores the future direction of the techniques introduced in this dissertation. These techniques have potential implications for understanding immune-related disorders and developing targeted therapies.